Treatment of congestive heart failure

ABSTRACT

A method of treating congestive heart failure (CHF) in a human patient comprises treating an aliquot of the patient&#39;s blood ex vivo with at least one stressor selected from the group consisting of a temperature above or below body temperature, an electromagnetic emission and an oxidative environment, followed by administering the aliquot of treated blood to the patient. The treatment can be used on its own or as an adjunctive therapy in combination with conventional CHF treatments.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to methods for treating congestive heart failure,in particular by the administration to a human subject of an aliquot ofmodified blood, optionally in combination with one or more othertreatments for alleviating the symptoms of congestive heart failure.

2. Description of the Prior Art

Congestive heart failure (CHF) is a relatively common disorder affectingapproximately five million Americans, with a mortality rate of over80,000 per year. It is believed that CHF is not a distinct diseaseprocess in itself, but rather represents the effect of multipleanatomic, functional and biologic abnormalities which interact togetherto ultimately produce progressive loss of the ability of the heart tofulfill its function as a circulatory pump.

CHF may be caused by the occurrence of an index event such as amyocardial infarction (heart attack) or be secondary to other causessuch as hypertension or cardiac malformations such as valvular disease.The index event or other causes result in an initial decline in thepumping capacity of the heart, for example by damaging the heart muscle.This decline in pumping capacity may not be immediately noticeable, dueto the activation of one or more compensatory mechanisms. However, theprogression of CHF has been found to be independent of the patient'shemodynamic status. Therefore, the damaging changes caused by thedisease are present and ongoing even while the patient remainsasymptomatic. In fact, the compensatory mechanisms which maintain normalcardiovascular function during the early phases of CHF may actuallycontribute to progression of the disease, for example by exertingdeleterious effects on the heart and circulation.

Some of the more important pathophysiologic changes which occur in CHFare activation of the hypothalamic-pituitary-adrenal axis, systemicendothelial dysfunction and myocardial remodeling.

Therapies specifically directed at counteracting the activation of thehypothalamic-pituitary-adrenal axis include beta-adrenergic blockingagents (β-blockers), angiotensin converting enzyme (ACE) inhibitors,certain calcium channel blockers, nitrates and endothelin-1 blockingagents. Calcium channel blockers and nitrates, while producing clinicalimprovement have not been clearly shown to prolong survival whereasβ-blockers and ACE inhibitors have been shown to significantly prolongfife, as have aldosterone antagonists. Experimental studies usingendothelin-1 blocking agents have shown a beneficial effect.

Systemic endothelial dysfunction is a well-recognized feature of CHF andis clearly present by the time signs of left ventricular dysfunction arepresent. Endothelial dysfunction is important with respect to theintimate relationship of the myocardial microcirculation with cardiacmyocytes. The evidence suggests that microvascular dysfunctioncontributes significantly to myocyte dysfunction and the morphologicalchanges which lead to progressive myocardial failure.

In terms of underlying pathophysiology, evidence suggests thatendothelial dysfunction may be caused by a relative lack of NO which canbe attributed to an increase in vascular O₂ ⁻ formation by anNADH-dependent oxidase and subsequent excess scavenging of NO. Potentialcontributing factors to increased O₂ ⁻ production include increasedsympathetic tone, norepinephrine, angiotensin II, endothelin-1 andTNF-α. In addition, levels of IL-10, a key anti-inflammatory cytokine,are inappropriately low in relation to TNF-α levels. It is now believedthat elevated levels of TNF-α, with associated proinflammatory cytokinesincluding IL-6, and soluble TNF-α receptors, play a significant role inthe evolution of CHF by causing decreased myocardial contractility,biventricular dilatation, and hypotension and are probably involved inendothelial activation and dysfunction. It is also believed that TNF-αmay play a role in the hitherto unexplained muscular wasting whichoccurs in severe CHF patients. Preliminary studies in small numbers ofpatients with soluble TNF-receptor therapy have indicated improvementsin NYHA functional classification and in patient well-being, as measuredby quality of life indices.

Myocardial remodeling is a complex process which accompanies thetransition from asymptomatic to symptomatic heart failure, and may bedescribed as a series of adaptive changes within the myocardium. Themain components of myocardial remodeling are alterations in myocytebiology, loss of myocytes by necrosis or apoptosis, alterations in theextracellular matrix and alterations in left ventricular chambergeometry. It is unclear whether myocardial remodeling is simply theend-organ response that occurs following years of exposure to the toxiceffects of long-term neurohormonal stimulation, or whether myocardialremodeling contributes independently to the progression of heartfailure. Evidence to date suggests that appropriate therapy can slow orhalt progression of myocardial remodeling.

Although presently used treatments can alleviate symptoms of CHF andcorrect certain pathophysiologic abnormalities caused by the diseaseprocess, CHF remains a relentlessly progressive condition with arelatively high rate of mortality. In fact, relative reductions inmorbidity and mortality brought about by existing drugs are on the orderof about 10 to 25 percent Therefore, the need exists for additive orsuperior treatments for CHF, especially those which can significantlymodify the underlying disease.

SUMMARY OF THE INVENTION

The present invention overcomes at least some of the above-noted andother disadvantages of presently known CHF therapies by providing amethod for treating CHF in which an aliquot of mammalian blood istreated ex vivo and subsequently introduced into the body of a mammaliansubject.

The aliquot of blood is treated by being subjected to one or morestressors which have been found to modify the blood. According to thepresent invention, the blood aliquot can be modified by subjecting theblood, or separated cellular or non-cellular fractions of the blood, ormixtures of the separated cells and/or noncellular fractions of theblood, to stressors selected from temperature stressors, electromagneticemissions and oxidative environments, or any combination of suchstressors, simultaneously or sequentially.

As discussed above, the pathophysiologic changes associated with CHFinclude immune activation, endothelial dysfunction and loss of myocytesthrough necrosis and/or apoptosis. The treatment method of the presentinvention has been shown to produce therapeutic benefits in each ofthese three areas.

With respect to immune activation, the treatment of the presentinvention has been found to modulate levels of inflammatory cytokines inseveral Th1/TNF-α-dependent experimental inflammatory models indifferent species. For example, the treatment has been shown to reduceallergic contact hypersensitivity in Balb/c mice, a Th1-driven immunereaction mediated by TNF-α (Shivji et al., Journal of Cutaneous Medicineand Surgery 4: 132-137, 2000); to down-regulate expression of IL6 mRNAin adjuvant-induced arthritis in the Lewis rat model of inflammatorydisease; and to decrease the proportion of Th1 to Th2 cells in patientswith scleroderma, a Th1-driven autoimmune disease (Rabinovich et al.,Poster presented at the XII Pan-American Congress of Rheumatology,Montreal, Canada, Jun. 21-25, 1998). It is believed that the treatmentdown-regulates the pro-inflammatory Th1-type immune response, forexample by increasing anti-inflammatory TH2-type cytokines, includingIL-10.

The treatment of the invention has been found to improve endothelialfunction in a number of studies conducted in humans and in animals. Forexample, the treatment has been found to improve endothelial-dependentvasodilator function in an open study on patients with severe primaryRaynaud's disease (Cooke et al., International Journal of Angiology 16:250-254, 1997), to improve the rate of recovery of skin blood flowfollowing temporary occlusion in a double-blind, placebo-controlledstudy in patients with advanced peripheral vascular disease secondary toatheroscierosis (Courtman et al., Circulation Vol 102, #18, suppl II,2000), to reduce progression of atherosclerosis in the cholesterol-fedLDD receptor deficient mouse (Babaei et al., Journal of the AmericanCollege of Cardiology 35 (Suppl. A): 243, 1999), and to markedly improveendothelial-dependent vasodilator function to acetylcholine in severelyatherosclerotic, hypercholesterolemic Watanabe rabbits as evidenced byan increased vasodilatory response to the nitric oxide agonist(acetylcholine) (Courtman et al., above). It is believed that theimprovement in endothelial function is due to an anti-inflammatoryeffect and to increased availability of NO which may result in animprovement in vasodilatory capacity, known to be severely impaired inCHF patients.

With regard to myocyte loss, the method of the invention is believed todecrease levels of apoptosis and necrosis. It has been shown that thetreatment can protect the kidney from ischemia/reperfusion (I/R) damageknown to be associated with increased apoptotic cell death (Tremblay etal., Pathophysiology 5:26; Chen et al., Médecline Sciences 15 (Suppl.1): 16), and can reduce apoptosis in the kidney following I/R asdetermined by DNA laddering and density of apoptotic nuclei stained byTdt.

Because the treatment of the invention produces therapeutic benefits inthree areas in which pathophysiologic changes occur in CHF, namelyendothelial dysfunction, production of inflammatory cytokines andmyocyte loss due to apoptosis, there is provided a strong theoreticalbasis on which to predict that the treatment of the invention would bebeneficial to patients with CHF. The method of the invention may be usedas a CHF therapy on its own or in combination with other therapies, suchas nitrate therapy, β-blockers, ACE inhibitors, AT receptor blockingagents, aldosterone antagonists, calcium channel blocking agents, TNFblocking agents, suppressors of production of TNF-α, and/or other moreroutine treatment measures such as sodium and fluid restriction,diuretics, digitalis, etc. Specific drugs known to suppress TNF-αproduction include pentoxifylline, amrinone, adenosine, thalidomide, TNFconverting enzyme (TACE) inhibitors and dexamethasone. Specific TNFblocking agents include monoclonal antibodies and etanercept.

Accordingly, in one aspect the present invention provides a method oftreating CHF in a human patient suffering therefrom, comprising: (a)treating an aliquot of the patient's blood ex vivo with at least onestressor selected from the group consisting of a temperature above orbelow body temperature, an electromagnetic emission and an oxidativeenvironment; and (b) administering the aliquot of blood treated in step(a) to the patient, wherein the aliquot has a volume sufficient toalleviate CHF in the patient.

In another aspect, the present-invention provides a combinationtreatment for CHF in a human patient suffering therefrom, thecombination treatment including the administration to the patient of analiquot of the patient's own blood which has been treated ex vivo withone or more stressors selected from an oxidative environment, thermalstress and electromagnetic emission, and a treatment selected from thegroup consisting of nitrates, β-blockers, ACE inhibitors, AT receptorblocking agents, aldosterone antagonists, calcium channel blockingagents, TNF blocking agents, suppressors of production of TNF-α, sodiumand fluid restriction, diuretics and digitalis.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is now described, by way of example only, with referenceto the accompanying drawings in which:

FIGS. 1 and 2 of the accompanying drawings are graphical presentationsof the results obtained from Example 2 described below;

FIG. 3 of the accompanying drawings is a graphical presentation of theresults obtained from Example 3 described below;

FIG. 4 of the accompanying drawings is a graphical presentation of theresults obtained from Example 4 described below;

FIGS. 5 to 8 of the accompanying drawings are graphical presentations ofthe, results obtained from Example 5 described below; and

FIG. 9 of the accompanying drawings is a graphical presentation of theeffects of the treatment of the invention in contact hypersensitivityTh1-mediated inflammation

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

According to a preferred process of the present invention, an aliquot ofblood is extracted from a mammalian subject, preferably a human, and thealiquot of blood is treated ex vivo with certain stressors, described inmore detail below. The terms “aliquot”, “aliquot of blood” or similarterms used herein include whole blood, separated cellular fractions ofthe blood including platelets, separated non-cellular fractions of theblood including plasma, and combinations thereof. The effect of thestressors is to modify the blood, and/or the cellular or non-cellularfractions thereof, contained in the aliquot. The modified aliquot isthen re-introduced into the subject's body by any route suitable forvaccination, preferably selected from intra-arterial injection,intramuscular injection, intravenous injection, subcutaneous injection,intraperitoneal injection, and oral, nasal or rectal administration.

The stressors to which the aliquot of blood is subjected ex vivoaccording to the method of the present invention are selected fromtemperature stress (blood temperature above or below body temperature),an oxidative environment and an electromagnetic emission, individuallyor in any combination, simultaneously or sequentially. Suitably, inhuman subjects, the aliquot has a volume sufficient that, whenre-introduced into the subject's body, at least partial alleviation ofCHF is achieved in the subject. Preferably, the volume of the aliquot isup to about 400 ml, preferably from about 0.1 to about 100 ml, morepreferably from about 5 to about 15 ml, even more preferably from about8 to about 12 ml, and most preferably about 10 ml, along with ananticoagulant, e.g. 2 ml sodium citrate.

It is preferred, according to the invention, to apply all three of theaforementioned stressors simultaneously to the aliquot under treatment,in order to ensure the appropriate modification to the blood. It mayalso be preferred in some embodiments of the invention to apply any twoof the above stressors, for example to apply temperature stress andoxidative stress, temperature stress and an electromagnetic emission, oran electromagnetic emission and oxidative stress. Care must be taken toutilize an appropriate level of the stressors to thereby effectivelymodify the blood to alleviate CHF in the subject.

The temperature stressor warms the aliquot being treated to atemperature above normal body temperature or cools the aliquot belownormal body temperature. The temperature is selected so that thetemperature stressor does not cause excessive hemolysis in the bloodcontained in the aliquot and so that, when the treated aliquot isinjected into a subject, alleviation of CHF will be achieved.Preferably, the temperature stressor is applied so that the temperatureof all or a part of the aliquot is up to about 55° C., and morepreferably in the range of from about −5° C. to about 55° C.

In some preferred embodiments of the invention, the temperature of thealiquot is raised above normal body temperature, such that the meantemperature of the aliquot does not exceed a temperature of about 55°C., more preferably from about 40° C. to about 50° C., even morepreferably from about 40° C. to about 44° C., and most preferably about42.5±1° C.

In other preferred embodiments, the aliquot is cooled below normal bodytemperature such that the mean temperature of the aliquot is within therange of from about −5° C. to about 36.5° C., more preferably from about10° C. to about 30° C., and even more preferably from about 15° C. toabout 25° C.

The oxidative environment stressor can be the application to the aliquotof solid, liquid or gaseous oxidizing agents. Preferably, it involvesexposing the aliquot to a mixture of medical grade oxygen and ozone gas,most preferably by bubbling through the aliquot, at the aforementionedtemperature range, a stream of medical grade oxygen gas having ozone asa minor component therein. The ozone content of the gas stream and theflow rate of the gas stream are preferably selected such that the amountof ozone introduced to the blood aliquot, either on its own or incombination with other stressors, does not give rise to excessive levelsof cell damage such that the therapy is rendered ineffective. Suitably,the gas stream has an ozone content of up to about 300 μg/ml, preferablyup to about 100 μg/ml, more preferably about 30 μg/ml, even morepreferably up to about 20 μg/ml, particularly preferably from about 10μg/ml to about 20 μg/ml, and most preferably about 14.5±1.0 μg/ml. Thegas stream is suitably supplied to the aliquot at a rate of up to about2.0 litres/min, preferably up to about 0.5 litres/min, more preferablyup to about 0.4 litres/min, even more preferably up to about 0.33litres/min, and most preferably about 0.24±0.024 litres/min, at STP. Thelower limit of the flow rate of the gas stream is preferably not lowerthan 0.01 litres/min, more preferably not lower than 0.1 litres/min, andeven more preferably not lower than 0.2 litres/min.

The electromagnetic emission stressor is suitably applied by irradiatingthe aliquot under treatment from a source of an electromagnetic emissionwhile the aliquot is maintained at the aforementioned temperature andwhile the oxygen/ozone gaseous mixture is being bubbled through thealiquot Preferred electromagnetic emissions are selected from photonicradiation, more preferably UV, visible and infrared light, and even morepreferably UV light. The most preferred UV sources are UV lamps emittingprimarily UV-C band wavelengths, i.e. at wavelengths shorter than about280 nm. Such lamps may also emit amounts of visible and infrared light.Ultraviolet light corresponding to standard UV-A (wavelengths from about315 to about 400 nm) and UV-B (wavelengths from about 280 to about 315)sources can also be used. For example, an appropriate dosage of such UVlight, applied simultaneously with the aforementioned temperature andoxidative environment stressors, can be obtained from up to eight lampsarranged to surround the sample container holding the aliquot, operatedat an intensity to deliver a total UV light energy at the surface of theblood of from about 0.025 to about 10 joules/cm², preferably from about0.1 to about 3.0 joules/cm^(2,) may advantageously be used. Preferably,four such lamps are used.

The time for which the aliquot is subjected to the stressors is normallywithin the time range of up to about 60 minutes. The time depends tosome extent upon the chosen intensity of the electromagnetic emission,the temperature, the concentration of the oxidizing agent and the rateat which it is supplied to the aliquot. Some experimentation toestablish optimum times may be necessary on the part of the operator,once the other stressor levels have been set. Under most stressorconditions, preferred times will be in the approximate range of fromabout 2 to about 5 minutes, more preferably about 3 or about 3½ minutes.The starting blood temperature, and the rate at which it can be warmedor cooled to a predetermined temperature, tends to vary from subject tosubject. Such a treatment provides a modified blood aliquot which isready for injection into the subject.

In the practice of the preferred process of the present invention, theblood aliquot may be treated with the stressors using an apparatus ofthe type described in U.S. Pat. No. 4,968,463 to Mueller. The aliquot isplaced in a suitable, sterile, UV light-transmissive container, which isfitted into the machine. The UV lamps are switched on for a fixed periodbefore the gas flow is applied to the aliquot providing the oxidativestress, to allow the output of the UV lamps to stabilize. The UV lampsare typically on while the temperature of the aliquot is adjusted to thepredetermined value. e.g. 42.5±1° C. Then the oxygen/ozone gas mixture,of known composition and controlled flow rate, is applied to thealiquot, for the predetermined duration of up to about 60 minutes,preferably 2 to 5 minutes and most preferably about 3 minutes asdiscussed above, so that the aliquot experiences all three stressorssimultaneously. In this way, blood is appropriately modified accordingto the present invention to achieve the desired effects.

A subject preferably undergoes a course of treatments, each individualtreatment comprising removal of a blood aliquot, treatment thereof asdescribed above and re-administration of the treated aliquot to thesubject. A course of such treatments may comprise daily administrationof treated blood aliquots for a number of consecutive days, or maycomprise a first course of daily treatments for a designated period oftime, followed by an interval and then one or more additional courses ofdaily treatments.

In one preferred embodiment, the subject is given an initial course oftreatments comprising the administration of 4 to 6 aliquots of treatedblood. In another preferred embodiment, the subject is given an initialcourse of therapy comprising administration of from 2 to 4 aliquots oftreated blood, with the administration of any pair of consecutivealiquots being either on consecutive days, or being separated by a restperiod of from 1 to 21 days on which no aliquots are administered to thepatient, the rest period separating one selected pair of consecutivealiquots being from about 3 to 15 days. In a more specific, preferredembodiment, the dosage regimen of the initial course of treatmentscomprises a total of three aliquots, with the first and second aliquotsbeing administered on consecutive days and a rest period of 11 daysbeing provided between the administration of the second and thirdaliquots. In the method of the invention, it is preferred that no morethan one aliquot is administered to the subject on any given day.

It may be preferred to subsequently administer additional courses oftreatments following the initial course of treatments. Preferably,subsequent courses of treatments are administered at least about threeweeks after the end of the initial course of treatments. In oneparticularly preferred embodiment, the subject receives a second courseof treatments comprising the administration of one aliquot of treatedblood every 30 days following the end of the initial course oftreatments, for a period of 6 months.

It will be appreciated that the spacing between successive courses oftreatments should be such that the positive effects of the treatment ofthe invention are maintained, and may be determined on the basis of theobserved response of individual subjects.

As discussed above, the method of the present invention may preferablybe used as an adjunctive treatment in combination with other therapiesfor CHF. Preferred examples of such other therapies include one or moreof ACE inhibitors, β-blockers, aldosterone antagonists, TNF blockers,suppressors of TNF production and other forms of routine therapy.

The invention is further illustrated and described with reference to thefollowing specific examples.

EXAMPLE 1

This example describes a study conducted to determine the effect of thetreatment of the invention on endothelial function in Watanabe rabbits,known to develop complex atherosclerotic lesions during the first yearof life. As previously mentioned, endothelial dysfunction is linked tothe pathophysiology of CHF.

The rabbits entered the study at 7 to 8 months of age, and wererandomized into three groups, a first group to be sacrificed immediatelyfor baseline measurements, a second group (n=10) which receivedinjections of blood treated according to the invention, and a thirdgroup (n=10) which received sham treatments comprising injections ofuntreated blood.

The treatment comprised a total of 4 injections of treated blood over aperiod of 10 weeks. The blood was treated by exposure to the followingthree stressors in an apparatus as generally described in U.S. Pat. No.4,968,483 to Mueller et al.:

-   -   (a) an elevated temperature of 42.5° C.±1.0° C.;    -   (b) a gas mixture of medical grade oxygen containing 14.5±1.0        μg/ml of ozone, bubbled through the blood at a flow rate of        240±24 ml/min for 3 minutes; and    -   (c) ultraviolet light at a wavelength of 253.7 nm, and a total        energy density of 2.0 joules/cm² (with some fluctuation within        the previously mentioned range).

The treated blood was administered to the animals by intra-muscularinjection. The control animals were administered intra-muscularinjections of untreated blood on the same injection schedule as thetreated animals.

All animals were sacrificed at 11 months of age. Ring preparations weretaken from the iliac arteries of the animals and were evaluated for theamount of relaxation induced by acetylcholine (an endothelial-dependentvasodilator) after being treated with phenylephrine (a vasoconstrictor).

Evaluation of the ring preparations showed a significant increase inendothelial-mediated vasorelaxation (52.2±6%) was observed in thetreated animals as compared to the control animals injected withuntreated blood (22.9±4%, p less than 0.001).

No relaxation was observed when the endothelium was removed from thering preparations, further confirming the endothelium-specific effect ofthe treatment of the invention.

EXAMPLE2

This example describes a study into the effects of the treatment of theinvention therapy on patients suffering from peripheral vascular disease(PVD). The study was conducted at the University Hospital, Lund, Sweden.

The study comprised a placebo-controlled, double blind study in 18patients (7 males, 11 females) with moderately advanced PVD, whose mainsymptom was intermittent claudication. The patients participating in thestudy were recruited from the attending population of the Department ofInternal Medicine of the University Hospital, Lund, Sweden.

The patients were randomly assigned to receive either placebo(intramuscular injection of 10 ml warm saline) or treatment according tothe invention comprising intramuscular injections of 10 ml of treatedautologous blood. The treatment of the blood involved the collection ofa 10 ml aliquot of a patient's venous blood into 2 ml of sodium citrate3-4% as anticoagulant. Each blood aliquot was transferred to a sterile,disposable low-density polyethylene vessel and then exposed to thefollowing conditions in an apparatus as generally described in U.S. Pat.No. 4,968,483 to Mueller et al.:

-   -   (d) elevated temperature of 42.5° C.±1.0° C.;    -   (e) medical oxygen containing 14.5±1.0 ug/ml of ozone bubbled        through the blood aliquot at a flow rate of 240±24 ml/min at STP        for 3 minutes; and    -   (f) ultra-violet light at a wavelength of 253.7 nm, and a total        energy of about 2.0 joules/cm².

Each patient received a total of 12 injections of saline or treatedblood over a period of 9 weeks.

The therapy was assessed by measuring the recovery rate of skin bloodflow and oxygen tension following total temporary occlusion of bloodflow in the extremities of each patient prior to commencement of thetherapy and at 3 weeks, 6 weeks, 9 weeks and 2 months following theinitiation of the therapy.

Skin blood flow in the foot was measured by Laser Doppler Fluxmetry(LDF) and oxygen tension was determined by measurement of transcutaneousskin oxygen pressure (TcpO₂) in the foot. In patients receiving thetreatment of the invention, a strong trend was observed toward atreatment-related reduction in both the total time to reach maximumperfusion (TP_(H)) and the halftime to reach maximum perfusion(T_(1/2)P_(H)) indicative of an improvement in the rate of recovery ofskin blood flow. No change was observed in the control group.

The improved rate of recovery of blood flow in patients treatedaccording to the invention was apparent during the course of treatmentsand persistent throughout, but did not reach significance until 2 monthsfollowing initiation of the therapy. A comparison of the T_(1/2)P_(H)for the placebo and treated groups, as measured by LDF, is shown in FIG.1.

There was also an observed trend toward more rapid recovery of skinoxygen content in the treated group. This difference becamestatistically significant at 2 months following the initiation of thetherapy. A comparison of the half-time to maximum TcpO₂ after ischemia(O₂T_(1/2)) for the treated group compared to the placebo group is shownin FIG. 2.

The study therefore demonstrated that, in this group of moderatelyadvanced PVD patients, the treatment of the invention had a clearbiological effect on the rate at which blood flow in the skin of thefoot was recovered following a period of total occlusion ischemia. Asimilar effect, but of smaller magnitude, was noted for the rate ofTcpO₂ recovery, whereas patients receiving placebo treatment showed nochange. These results suggest that the treatment of the invention has abeneficial effect on endothelial function, and appears to improve skinmicrocirculatory function in patients with PVD.

EXAMPLE 3

This example relates to the use of the treatment of the invention toprevent the onset of arthritis, and describes the results of a studyconducted in an established animal model of arthritis. The specificanimal model used in this study was adjuvant-induced arthritis in rats(see, for example, Pearson, C., 1956, “Development of Arthritis,periarthritis and periostitis in rats given adjuvant”, Proc. Soc. Exp.Biol. Med., 91:95). According to this model, arthritis is induced inrats by injecting them with adjuvant containing Mycobacterium butyricum.

Male Lewis rats, 4 to 5 weeks of age, 100 to 120 g, were obtained fromCharles River Laboratories, quarantined one week and entered into thestudy. An adjuvant mixture was prepared for induction of arthritis bysuspending 50 mg M. butyricum (Difco Laboratories, Inc., Detroit, Mich.)in 5 ml light white paraffin oil—m3516 (Sigma Chemical Co., St. Louis,Mo.) and thoroughly mixed using a homogenizer. Aliquots of the mixturesufficient to supply 0.15 mg M. butyricum was injected into each animalsubcutaneously, at the base of the tail. Symptoms of arthritis appearedabout 12 days after induction, in each animal, as evidenced by limbswelling.

Two rats, which were not injected with the adjuvant mixture, were usedas blood donors. Blood was collected from the donors by cardiacpuncture, and 10 ml of citrated blood was transferred to a sterile, lowdensity polyethylene vessel for ex vivo treatment with stressorsaccording to the invention. Using an apparatus as generally described inthe above-mentioned Mueller patent, the blood was stressed by atreatment according to the invention.

Six animals were given a course of 2 injections of 0.2 ml aliquots ofthe treated blood, the injections being administered on consecutive daysafter the onset of arthritis. A control group of 8 rats receivedinjections of untreated blood using the same injection schedule as thetreated animals. Injections commenced one day after the induction ofarthritis. Hind paw volumes of the animals were measured, on alternatedays, after onset of arthritis, by water displacement in a 250 ml beakerusing a top-loaded Mettler balance. The results for each group ofanimals were averaged and are presented graphically on the accompanyingFIG. 3, a plot of mean foot volume against days after induction ofarthritis. The upper curve is derived from the control group of animals,the lower curve from the animals which received the course of injectionsof treated blood. A significant decrease in the severity of thearthritis, as indicated by lower foot volumes, is apparent for thetreated animals as compared to the animals of the control group.

The above results show that treatment of subjects with modifiedmammalian blood can effectively prevent the onset of arthritis inmammals.

The expression of IL-6 mRNA in lymph nodes of treated and untreatedanimals was measured 10 days after induction of arthritis, and theresults are presented below in Table I. TABLE 1 Treatment IL-6 copy no.(per 4500 actin units) Active <35 (n = 8) Control 254 ± 203 (n = 8)

The results shown above in Table I show that the treatment according tothe invention can modulate levels of inflammatory cytokines in aTh1/TNF-α-dependent model of arthritis. There is evidence thatproduction of inflammatory cytokines such as IL-6 and TNF-α is linked tothe pathophysiology of CHF.

EXAMPLE 4

The experiment reported in this example demonstrates, by use of ananimal model system involving ischemia and subsequent reperfusion ofvarious body organs, that the treatment of the present invention has theeffect of reducing apoptosis and necrosis. Ischemia-reperfusion injuriesare known to involve increase of apoptosis and necrosis in the affectedorgans and tissues—see for example Salkumar p, et at. “Mechanisms ofcell death in hypoxia/reoxygenation injury”, Oncogene Dec. 24, 1998;17(25):3341-9; and Burns A. T. et. al., “Apoptosis inischemia/reperfusion injury of human renal allografts”, Transplantation,Oct. 15, 1998; 66(7): 872-6, and other publications both preceding andfollowing those. Known techniques of determination of apoptosis at thecellular level are employed in this example.

Pure-bred normal beagle dogs, aged 1-2 years, equal numbers of males andfemales, were used as the experimental animals. The animals wereseparated into four groups, A, B, C and D, each group consisting of sixanimals, three males and three females. Animals of groups A and C weresubjected to the process of the invention, by being subjected to two10-day courses of daily removal of an 8 ml aliquot of blood,extracorporeal treatment of the aliquot with oxygen/ozone, UV radiationand heat, and re-administration of 5 ml of the treated aliquot to thesane animal, by intramuscular injection.

Each such treatment was conducted as follows.

An 8-ml aliquot of blood was extracted from the animal, treated withsodium crate (2 ml) and placed in a sterile container. It was subjectedsimultaneously to the UV radiation, oxygen/ozone gas oxidativeenvironment and elevated temperature stressors, in an apparatus asgenerally described in the aforementioned Mueller U.S. Pat. No.4,969,483. More specifically, the blood sample in the sterile,UV-transparent container was heated using infra-red lamps to 42.5° C.,and whilst being maintained at that temperature, it was subjected to UVradiation of predominant wavelength 253.7 nm under the preferredconditions previously described. Simultaneously, a mixture of medicalgrade oxygen and ozone, of ozone content 13.5-15.5 μg/ml was bubbledthrough the blood sample at a flow rate within the range from 60-240mls/min (STP). The time of simultaneous UV exposure and gas mixture feedwas 3 minutes. A 5 ml portion of the treated blood aliquot wasreinjected intramuscularly into each test animal.

Each animal of groups A and C, receiving the courses of treatmentaccording to the invention, experienced a three week rest period betweenthe 10-day courses of treatment. Groups B and D were the control groups,given two 10-day courses of daily injections of 5 ml of physiologicalsaline, with a three-week rest period between the 10-day courses.

One day following the second course of injections, the animals wereanaesthetized under light gas anaesthesia, and the right kidney of eachanimal was removed through a back incision. An occlusive dip was placedon the remaining renal artery and vein, to expose the left kidney totransient ischemia, for 60 minutes. Then the clip was removed to allowreperfusion of the kidney by normal blood flow.

The animals were observed for 6 days after the ischemia procedure, andthen sacrificed. The ischemic kidney of each animal was surgicallyremoved and divided into two parts. One part was kept frozen at −80° C.,and the other part was fixed in 10% formalin for immuno- and routinehistopathology studies.

Mitochondrial membrane potential was measured in proximal tubular cellsisolated from the ischemic and control kidneys, both at the time ofremoval of the control kidney and following sacrifice. For this purpose,dog kidney proximal tubes were purified from normal or ischemic kidneycortexes by the collagenase treatment procedure described by Marshanskyet. al., “Isolation of heavy endosomes from dog proximal tubes insuspension”, J. Membr. Biol 153(1), 59-73, 1996. Renal mitochondria wereisolated in suspension by differential centrifugation (see Marshansky,“Organic hydroperoxides at high concentrations cause energization andactivation of AATP synthesis in mitochondria”, J. Biol. Chem. 264(7),3670-3673, 1989, after tissue homogenization in a buffer containing 250mM sucrose, 10 mM HEPES-Tris (pH 7.5), and 250 μM EDTA. Cell debris wasremoved by centrifugation at 10,000 g for 30 minutes. The mitochondriawere washed with the sucrose/HEPES buffer without EDTA.

Mitochondrial membrane potential was measured as described by Kroemer,G., Zamzam, N. and Susin, S. A., “Mitochondrial control of apoptosis”,(Review) Immunology Today (1997) v. 18, p 44-51; with JC-1 dye—seeSalvioli et. al., “JC-1, but not DIOC6(3) or rhodamine 123, is areliable fluorescent probe to assess delta psi changes in intact cells:implications for studies on mitochondrial functionality duringapoptosis”, FEBS Letters 411 (1), 77-82, 1987. JC-1 fluorescence in thesuspension of purified mitochondria from normal and ischemic kidneys wasmonitored continuously on a Deltascan Model RFM-2001 spectrofluorimeter(Photon Technology International, South Brunswick, N.J.). The excitationwavelength was 490 nm (slit width 2 nm) and the emission wavelength was590 nm (slit width 4 nm). The signals were recorded using Felix®(Version 1.1) software. All measurements were performed with continuousstirring at 37° C. The incubation buffer for measurement ofmitochondrial membrane potential contained 200 mM sucrose, 5 mM MgCl₂, 5mM KH₂PO₄, 0.1 μM of JC-1 and 30 Mm HEPES-Tris (pH 7.5). Theconcentrations of the substrate and inhibitors were 10 mM succinate, 0.1μM rotenone with or without 0.1 μM FCCP. Proximal tubule mitochondrialmembrane potential was estimated in the right (control) kidney prior toischemia and in the left (ischemic) kidney after sacrifice of the dogson day 6 following ischemia and was estimated as difference of JC-1fluorescence after uncoupling of mitochondria with FCCP as shown in theaccompanying FIG. 4A. For each measurement, 50 μg protein of purifiedmaterial was used.

JC-1 fluorescence is proportional to the mitochondrial membranepotential. The contralateral nephrectomized kidney served as control. Asis clear from the FIG. 4B, the treatment process of the invention didnot modify the membrane potential of the non-ischemic control rightkidney (p=0.445 for treated vs saline). However, the ischemic kidney ofthe saline-injected animals showed significantly lower (p<0.05)fluorescence compared to the control kidney. The stress treatmentaccording to the invention prevented the uncoupling of mitochondriaduring ischemic/reperfusion, and membrane potential showed nosignificant difference (p=0.244) between ischemic and control kidneys.This parameter remained significantly higher (p=0.0006) vssaline-injected dogs) in the ischemic kidneys of dogs pretreatedaccording to the process of the invention for at least 6 dayspost-reperfusion.

These results indicate that the process of the invention effectsprotection of the kidney against apoptosis and/or accelerates recoveryat the mitochondrial level. Accordingly the process of the invention isindicated for pre-conditioning of the cells, tissues and organs of amammalian body against subsequently encountered factors which willnormally accelerate apoptosis.

Specifically, the preservation of mitochondrial membrane potentialevidences the capacity of the therapy to protect mitochondria, andthereby to precondition cells against apoptosis.

EXAMPLE 5

A group of 12 male SHR rats was treated with either injections of pooledblood stressed as described in Example 4 above, or, in control animals,with injections of saline. Since the blood from all of the animals ofthis genetic strain is identical, blood from one animal of this samestrain was treated by the process of the invention for administration tothe test animal. The blood was treated with sodium citrate asanticoagulant, and placed in a sterile container They received eitherinjections of 150 μl of stressed blood on days-14 and -13 followed by arest period of 11 days and a third injection the day before ischemicsurgery, or injections in parallel with saline. On the day of surgery,the rats were anaesthetized with light flurane, and the right kidney wasremoved through a mid-abdominal incision. The left kidney was thensubjected to transient ischemia by occlusion of the left renal arteryand vein using a micro-clip. The skin was then temporarily closed. After60 minutes of occlusion, the clip was removed and the wound was closedwith a suture. The animals were sacrificed 12 hours after reperfusion.

The ischemic and non-ischemic kidneys of the test animals were removedand subjected to DNA laddering tests. Oligonucleosomal DNA fragmentationinto 180 to 200 base pairs is a specific pattern which appears as aladder after agarose gel electrophoresis in various organs undergoingapoptosis. To estimate the degree of DNA fragmentation in the kidneycortex, an aliquot of pulverized kidney cortex was weighed and totaltissue DNA was extracted by the phenol-chloroform procedure after tissuedigestion with a proteinase K and RnaseA in the presence of EDTA. One μgof extracted DNA was labeled by enzymatic assay using terminaldeoxynucleotidyl transferase with P³²-dCTP (see Teiger et. al.,‘Apoptosis in pressure overload-induced heart hypertrophy in the rat’,J. Clin. Invest. 97, 2891-2897, 1996). Increasing quantities ofradio-labelled DNA were loaded onto 1.5% agarose gels. Afterelectrophoresis, DNA was transferred onto nylon membranes (Hybond) andthe radioactivity associated with 150 to 1500 bp DNA fragments wasquantified in a Phosphorimager (Molecular Dynamics). A regression linefor each sample was drawn for the radioactivity as a function of DNAloaded on the gel (see deBlois et. al., ‘Smooth muscle cell apoptosisduring vascular regression in spontaneously hypertensive rats.’Hypertension 29, 340-349, 1997). The slope of the linear regression lineserved as a DNA fragmentation index (cpm/pixel per μg DNA).

The results from ischemic-reperfused (I/R) kidneys and from normal,non-I/R kidneys, all from animals which did not receive injections ofstressed blood, are shown graphically on FIG. 5, a plot of the slope ofthe regression lines for the various samples (vertical axis) againsttime after initiation of reperfusion. The DNA laddering, indicative ofDNA fragmentation, was clearly increased in the ischemic kidney cortexcompared to the contralateral non-ischemic organ and the maximalattained at twelve hours returned to near basal values by 48 hours.Twelve hours was thus selected as the time point for study of the effectof the stressed blood of the invention on early ischemia-induced renalapoptosis.

FIG. 6A of the accompanying drawings is a picture of the electophoresisgel of the fragmented DNA, in the 150-1500 bp range, radio-labeled asdescribed to attach radioactivity labels to the DNA fragments. Trace Sderives from DNA of kidneys from animals which received salineinjections prior to kidney ischemia-reperfusion, and trace V derivesfrom DNA of kidneys of animals which received injections of the stressedblood prior to kidney ischemia-reperfusion. The Figure shows that 60minutes renal ischemia induced a clear accumulation of fragmented DNA inboth groups of rats at 12 h but the level of this parameter wassignificantly lower (p<0.05) in animals receiving the treated blood.FIG. 6B quantifies the amount of irradiation from the samples, inarbitrary units, and shows that DNA fragmentation-laddering occurs inboth S and V samples as a result of ischemia/reperfusion, but that theextent is markedly reduced in V samples as compared with S samples. Theresults presented on FIG. 68 are the means of six animals in each case.

These results confirm that the cytoprotective effect of theadministration of stressed blood according to the invention on renalreperfusion injury involves the inhibition of early or late apoptosis.

The ability of the treatment of the invention in reducing apoptosis inthe kidney following ischemia/reperfusion during the early phase ofapoptosis (after 12 hours) as determined by DNA laddering and density ofapoptotic nuclei stained by Tdt is shown in FIGS. 7 and 8, respectively.As well, FIG. 3B shows that cell numbers in the kidney followingischemia/reperfusion were also significantly higher in the animalstreated according to the invention.

EXAMPLE 6

This example describes the treatment of a small number of human patientswith advanced chronic congestive heart failure. The patients had NYHAclass III-IV chronic congestive heart failure, with a left ventricularejection fraction (LVEF) of less than 40% and a 6 minute walk distanceof less than 300 m. Some of the patients had previously received otherCHF treatments.

Protocol:

Patients receive a number of injections of treated blood. The treatmentschedule comprises injections on days 1, 2 and 14, followed by a singleinjection every 30 days for 5 months, each injection having a volume of10 ml. Each individual treatment comprises the following steps:

1. Collection of 10 ml of a patient's own venous blood into 2 ml of 3-4%sodium citrate for injection, USP. The sodium ctrate is added to thesample to prevent the blood from coagulating during the treatment.

2. Transfer of the citrated blood sample to a sterile, disposable,low-density polyethylene vessel.

3. Ex vivo treatment of the blood sample by simultaneous exposure to:

-   -   an elevated temperature of 42.6±1.0° C.,    -   a gas mixture of medical grade oxygen containing 14.5±1.0 μg/ml        of ozone which is bubbled through the blood sample at a flow        rate of 240±24 ml/min (at STP); and    -   ultraviolet light at a wavelength of 253.7 nm.

4. Transfer of the blood sample from the sterile disposable container toa sterile syringe.

5. Intramuscular injection of 2 ml or 10 ml of the treated blood sampleinto the gluteal muscle of the same patient, following a localanaesthetic (1 mL of 2% Novocain or equivalent) at the injection site.

The ex vivo treatment of the blood sample described in step (3) above isperformed with an apparatus as generally described in U.S. Pat. No.4,968,483 to Mueller et al. The blood sample is simultaneously exposedto all three stressors for a period of 3 minutes.

Assessment of CHF:

Patients are monitored for adverse events during each visit. As well, apost-treatment follow-up is conducted to monitor survival,hospitalizations, and significant adverse events.

The primary endpoints used to assess the effectiveness of the treatmentare changes in 6-minute walking distance and/or NYHA functionalclassification. Secondary endpoints include: improvement in cardiacfunction, reduction in diuretic requirement; reduction inhospitalization stay; and improvement in symptoms.

As demonstrated by the data described above, the treatment of thepresent invention has been shown to have significant biological activityin humans and in a number of animal model systems, all of which involveTh1/TNF-α dependent inflammatory responses. As mentioned above, it isbelieved that the treatment down-regulates the pro-inflammatory Th1-typeimmune response, for example by increasing anti-inflammatory TH2 typecytokines, including IL-10. This would at least partially explain theability of the treatment of the invention to produce therapeuticbenefits in each of the three areas which characterize CHF.

Furthermore, there is evidence to suggest that the treatment of theinvention is IL-10 dependent (FIG. 9 and Shahid S. et al., Journal ofInvestigative Dermatology, 14, No. 4, 2000), briging about anup-regulation of ant-inflammatory cytokines such as IL-10, and adown-regulation of TH-1 driven immune responses. It has also beenproposed that IL-10 may be an important component of the cytokinenetwork in CHF, as there appears to be a reduction in the level of IL-10in relation to TNF-α in CHF (Yamaoka et al., Jpn Circ J 63: 951-956).

Although the invention has been described with reference to specificpreferred embodiments, it will be appreciated that many variations maybe made to the invention without departing from the spirit or scopethereof. All such modifications are intended to be included within thescope of the following claims.

1-26. (canceled)
 27. A method of treating congestive heart failure (CHF)in a human patient suffering therefrom, comprising: (a) treating analiquot of the patient's blood ex vivo with a stressor comprising anoxidizing agent; and (b) administering the aliquot of blood step (a) tothe patient, wherein the aliquot has a volume sufficient to treat CHF inthe patient.
 28. The method of claim 27, wherein said stressor furthercomprises electromagnetic emissions and/or a temperature above or belowbody temperature.
 29. The method of claim 28, wherein all of thestressors are simultaneously administered to the aliquot.
 30. The methodof claim 28, wherein any two of the stressors are simultaneouslyadministered to the aliquot.
 31. The method of claim 28, wherein theelectromagnetic emission comprises ultraviolet light having one or moreUV-C band wavelengths.
 32. The method of claim 28, wherein thetemperature to which the aliquot is cooled or heated is a temperaturewhich does not result in substantial hemolysis of the blood in thealiquot.
 33. The method of claim 32, wherein the mean temperature of theblood in the aliquot is iii the range of from about 0° C. to about 36.5°C.
 34. The method of claim 32, wherein the mean temperature of the bloodin the aliquot is in the range of from about 10° C. to about 30° C. 35.The method of claim 32, wherein the temperature is in the range of fromabout 40° C. to about 50° C.
 36. The method of claim 35, wherein thetemperature is 42.5±1° C.
 37. The method of claim 27, wherein theoxidizing agent is introduced into the blood aliquot in an amount whichdoes not give rise to excessive levels of cell damage.
 38. The method ofclaim 27, wherein the volume of the aliquot is up to about 400 ml. 39.The method of claim 38, wherein the volume of the aliquot is about 10ml.
 40. The method of claim 38, wherein the volume of the aliquot isabout 2 ml.
 41. The method of claim 27, wherein the aliquot is subjectedto the stressor for a period of up to about 60 minutes.
 42. The methodof claim 41, wherein the aliquot is subjected to the stressor for aperiod of about 3 minutes.
 43. The method of claim 27, wherein the bloodis administered to the mammal by a method suitable for vaccinationselected from the group consisting of intra-arterial injection,intramuscular injection, intravenous injection, subcutaneous injection,intraperitoneal injection, and oral, nasal or rectal administration. 44.A combination treatment for congestive heart allure (CHF) in a humanpatient suffering therefrom, the combination treatment including theadministration to the patient of an aliquot of the patient's own bloodwhich has been treated selected from the group ex vivo with a stressorcomprising an oxidizing agent and selected from the group consisting ofnitrates, β-blockers, ACE inhibitors, AT receptor blocking agents,aldosterone antagonists, calcium channel blocking agents, TNF blockingagents, suppressors of production of TNF-α, sodium and fluidrestriction, diuretics and digitalis wherein the aliquot has a volumesufficient to treat CHF in the patient.
 45. The combination treatment ofclaim 44, wherein the suppressors of production of TNF-α are selectedfrom the group consisting of pentoxifyline, TACE inhibitors, amrinone,adenosine, thalidomide and dexamethasone.
 46. A method of treatingcongestive heart failure (CHF) in a human patient suffering therefrom,comprising: a) treating an aliquot of the patient's blood ex vivosimultaneously, for a time up to 60 minutes, with a combination ofstressors comprising (1) a mixture of ozone gas and medical gradeoxygen, the ozone gas being contained in the mixture in a concentrationof up to about 300 μg/ml; (2) ultraviolet light having one or more UV/Cband wavelengths; and (3) temperature in the range of from about 37° C.to about 55° C.; b) administering the aliquot of the treated in step a)to the patient, wherein the aliquot has a volume sufficient to treat theCHF in the patient.